DESCRIPTION: The long-term objectives of this proposal are to identify epithelial mechanisms that enable gastric mucosal cells to survive in the acid peptic environment. A primary canine gastric cell culture model was developed in which monolayers display resistance to apical acidification. Previous work established that the paracellular pathway is the primary site of resistance to H ions, and an early site of injury from excessive exposure to acid. Preliminary data show that endogenous growth factors decrease paracellular permeability and enhance tolerance to apical acid. EGF acts on apical and basolateral receptors whereas insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) act only through basolateral receptors. The proposed experiments will test the hypothesis that growth factor regulation of paracellular permeability involves rapid modulation of the apical junctional complex via activation of selective signal transduction pathways. Growth factor effects on paracellular permeability and barrier function, as measured by transepithelial resistance and by mannitol and inulin flux, will be compared. Addition of immunoneutralizing or receptor-blocking antibodies to either side will confirm the specificity of these effects and identify endogenous factors that regulate permeability. Growth factor-dependent modulation of tyrosine phosphorylation, apical/junctional distribution, and protein interactions among junctional components will be examined using immunoprecipitation/western blot analysis, confocal microscopy and co-immunoprecipitation. In addition, actin polymerization inhibitors will be used to assess the role of cytoskeletal regulation by growth factors. Lastly, activation and relevance of selected signaling molecules potentially mediating growth factor regulation of paracellular permeability and tyrosine phosphorylation (e.g., phosphatidylinositol-3 kinase or the Rho GTP binding proteins) will be assessed by use of specific inhibitors and activators. The studies should allow structural, functional and molecular characterization of the fundamental process of apical junctional regulation in a primary cell model with relevance to gastric epithelial physiology.